Docosahexaenoic Acid Increases Vesicular Glutamate Transporter 2 Protein Levels in Differentiated NG108-15 Cells.

Docosahexaenoic acid (DHA; 22:6n-3), which is enriched in the neuronal membrane, plays a variety of roles in the brain. Vesicular glutamate transporters (VGLUTs) are responsible for incorporating glutamine into synaptic vesicles. We investigated the influence of DHA on the fatty acid profile and the levels of VGLUT1 and VGLUT2 proteins in differentiated NG108-15 cells, a neuroblastoma-glioma hybrid cell line. NG108-15 cells were plated and 24 h later the medium was replaced with Dulbecco's modified Eagle's medium supplemented with 1% fetal bovine serum, 0.2 mM dibutyryl cAMP, and 100 nM dexamethasone, which was added to induce differentiation. After 6 d, the amount of DHA in the cells was increased by addition of DHA to the medium. VGLUT2 levels were increased by the addition of DHA. These data indicate that DHA affected the levels of VGLUT2 in NG108-15 cells under differentiation-promoting conditions, suggesting that DHA affects brain functions involving VGLUT2.

Comparison of the Physical Characteristics and Behavior in ABC Transporter A1, A7 or Apolipoprotein E Knockout Mice with Lipid Transport Dysfunction.

The physical characteristics and behavior of the ATP-binding cassette (ABC) A1, A7, and apolipoprotein (apo) E knockout (KO) mice with lipid transport dysfunction were investigated. These KO mice exhibited adequate growth, and their body masses increased steadily. No remarkable changes were observed in their blood pressure and heart rate. However, there was a slight increase in the heart rate of the ABCA7 KO mice compared with that of the wild-type (WT) mice. ABCA1 and apoE KO mice showed hypo- and hyper-cholesterol concentrations in the plasma, respectively. With regard to the cerebrum, however, the weight of the ABCA1 KO mice was lighter than those of the other genotypes. Furthermore, the cholesterol, triglyceride and phospholipid concentrations, and fatty acid composition were generally similar. Compared with the WT mice, ABCA1 KO mice stayed for a shorter time in the closed arm of the elevated plus maze, and performed worse in the initial stage of the Morris water maze. To thermal stimuli, the ABCA1 and apoE KO mice showed hyper- and hypo-sensitivities, respectively. Only the response of the ABCA1 KO mice was significantly inhibited by pretreatment with indomethacin. A low concentration of the prostaglandin E metabolites was detected in the plasma of the ABCA1 KO mice. Thus, ABCA1 is thought to play a specific role in the neural function.

Fully hydrogenated canola oil extends lifespan in stroke-prone spontaneously hypertensive rats.

BACKGROUND: Canola oil (Can) and several vegetable oils shorten the lifespan of stroke-prone spontaneously hypertensive rats (SHRSP). Although similar lifespan shortening has been reported for partially hydrogenated Can, the efficacy of fully hydrogenated oils on the lifespan remains unknown. The present study aimed to investigate the lifespan of SHRSP fed diets containing 10 % (w/w) of fully hydrogenated Can (FHCO) or other oils. METHODS: Survival test: Upon weaning, male SHRSP were fed a basal diet for rodents mixed with one of the test oils -i.e., FHCO, Can, lard (Lrd), and palm oil (Plm) throughout the experiment. The animals could freely access the diet and drinking water (water containing 1 % NaCl), and their body weight, food intake, and lifespan were recorded. Biochemical analysis test: Male SHRSP were fed a test diet with either FHCO, Can, or soybean oil (Soy) under the same condition, except to emphasize effects of fat, that no NaCl loading was applied. Soy was used as a fat source in the basal diet and was set the control group. Blood pressures was checked every 2 weeks, and serum fat levels and histological analyses of the brain and kidney were examined after 7 or 12 weeks of feeding. RESULTS: During the survival study period, the food consumption of FHCO-fed rats significantly increased (15-20 % w/w) compared with that of rats fed any other oil. However, the body weight gain in the FHCO group was significantly less (10-12 %) than that in the control group at 9-11 weeks old. The FHCO (> 180 days) intervention had the greatest effect on lifespan, followed by the Lrd (115 ± 6 days), Plm (101 ± 2 days), and Can (94 ± 3 days) diets. FHCO remarkably decreased the serum cholesterol level compared with Can and the systolic blood pressure from 12 to 16 weeks of age. In addition, while some rats in the Can group exhibited brain hemorrhaging and renal dysfunction at 16 weeks old, no symptoms were observed in the FHCO group. CONCLUSION: This current study suggests that complete hydrogenation decreases the toxicity of Can and even prolongs the lifespan in SHRSP.

Effects of arachidonic acid intake on inflammatory reactions in dextran sodium sulphate-induced colitis in rats.

The aim of this study was to investigate the effects of the administration of oral arachidonic acid (AA) in rats with or without dextran sulphate sodium (DSS)-induced inflammatory bowel disease. Male Wistar rats were administered AA at 0, 5, 35 or 240 mg/kg daily by gavage for 8 weeks. Inflammatory bowel disease was induced by replacing drinking water with 3 % DSS solution during the last 7 d of the AA dosing period. These animals passed loose stools, diarrhoea and red-stained faeces. Cyclo-oxygenase-2 concentration and myeloperoxidase activity in the colonic tissue were significantly increased in the animals given AA at 240 mg/kg compared with the animals given AA at 0 mg/kg. Thromboxane B2 concentration in the medium of cultured colonic mucosae isolated from these groups was found to be dose-dependently increased by AA, and the increase was significant at 35 and 240 mg/kg. Leukotriene B4 concentration was also significantly increased and saturated at 5 mg/kg. In addition, AA at 240 mg/kg promoted DSS-induced colonic mucosal oedema with macrophage infiltration. In contrast, administration of AA for 8 weeks, even at 240 mg/kg, showed no effects on the normal rats. These results suggest that in rats with bowel disease AA metabolism is affected by oral AA, even at 5 mg/kg per d, and that excessive AA may aggravate inflammation, whereas AA shows no effects in rats without inflammatory bowel disease.

Long-term high-soybean oil feeding alters regulation of body temperature in rats.

We investigated whether body temperature (BT) regulatory mechanisms are influenced by dietary fatty acids (FA). Male Wistar rats were fed a high-fat diet containing fish oil (HFD), soybean oil (HSD) or lard (HLD). At the 20-week intervention, the BT of the HSD and HLD groups were lower than that of the normal diet (ND) group in the light and dark periods. The intracerebroventricular injections of interleukin-1ß and bombesin in the HSD group induced greater hyperthermia and weaker hypothermia, respectively, than in the ND group. The HSD differentially affected BT under both physiological and pharmacological conditions. In the hypothalamus, the ratio of n-6/n-3 FAs was higher in the HSD group compared with the ND group. DNA microarrays revealed increased expression of thyroid-stimulating hormone ß-subunit, and decreased expression of several genes in the hypothalamus of the HSD group compared with the ND group. The HSD feeding increased several adipokine concentrations in the plasma. However, there were no adipokines or gene expressions that changed in only the HSD and HLD groups showing significant hypothermia under the physiological condition. These findings suggested that long-term HSD intake produces abnormal BT regulation. It is less likely that adipokines or proteins/peptides are involved in abnormal BT regulation under the physiological conditions after HSD feeding.

Docosahexaenoic acid contributes to increased CaMKII protein expression and a tendency to increase nNOS protein expression in differentiated NG108-15 cells.

Docosahexaenoic acid (DHA; 22:6n-3), an n-3 polyunsaturated fatty acid, has various important roles in brain functions. Nitric oxide (NO) produced by neuronal NO synthase (nNOS) and Ca2+/calmodulindependent protein kinase II (CaMKII) is also involved in brain functions. We investigated the influence of DHA on nNOS and CaMKII protein expression in differentiated NG108-15 cells. NG108-15 cells were seeded in 12-well plates, and after 24 h, the medium was replaced with Dulbecco's modified Eagle's medium containing 1% fetal bovine serum, 0.2 mM dibutyryl cyclic adenosine monophosphate and 100 nM dexamethasone as differentiation-inducing medium. When cells were cultured in differentiation-inducing medium, neurite-like outgrowths were observed on days 5 and 6. However, no significant difference in morphology was observed in cells with or without DHA treatment. With or without DHA addition, nNOS protein expression was increased on days 5 and 6 compared with day 0. This increase tended to be enhanced by DHA. CaMKII protein expression did not change after differentiation without DHA, but was significantly increased on day 6 compared with day 0 with DHA addition. These data indicate that DHA is involved in brain functions by regulating CaMKII and nNOS protein expression.

PMA induces GCMa phosphorylation and alters its stability via the PKC- and ERK-dependent pathway.

The glial cells missing a (GCMa) transcription factor plays a pivotal role in the placental development by regulating the expression of several genes in the placenta that are responsible for the proper formation of the syncytiotrophoblast. It is well known that the function of GCMa is regulated at both transcriptional and post-translational levels by the cyclic AMP (cAMP)/protein kinase A (PKA)-dependent pathway, the activation of which increases the GCMa protein level and leads to trophoblast differentiation into the syncytiotrophoblast. However, little is known about the regulatory control of GCMa by PKC-dependent signaling mechanism(s). To investigate whether GCMa is regulated by PKC-dependent pathway, we treated the human choriocarcinoma JEG-3 cells with phorbol 12-myristate 13-acetate (PMA) and studied its effect on the GCMa protein using a monoclonal anti-GCMa antibody we prepared. PMA caused a transient decrease in the endogenous GCMa protein level in JEG-3 cells that was accompanied by an increase in GCMa phosphorylation. The phosphorylation and degradation of GCMa by PMA treatment was effectively reduced by pretreatment with protein kinase C (PKC) inhibitors and a mitogen-activated protein kinase (MAPK) kinase (MEK) inhibitor, indicating a PKC- and MEK-dependent mechanism. Furthermore, we identified the serine residues 328, 378 and 383 to be the phosphorylation sites on GCMa that are involved in the PMA-induced degradation of GCMa. Our data demonstrate for the first time that GCMa is phosphorylated by the PKC- and MEK/extracellular signal-regulated kinase (ERK)-dependent mechanism, and that this phosphorylation is involved in its degradation process.

Proteomic analysis of survival of Rhodococcus jostii RHA1 during carbon starvation.

Rhodococcus jostii RHA1, a catabolically diverse soil actinomycete, is highly resistant to long-term nutrient starvation. After 2 years of carbon starvation, 10% of the bacterial culture remained viable. To study the molecular basis of such resistance, we monitored the abundance of about 1,600 cytosolic proteins during a 2-week period of carbon source (benzoate) starvation. Hierarchical cluster analysis elucidated 17 major protein clusters and showed that most changes occurred during transition to stationary phase. We identified 196 proteins. A decrease in benzoate catabolic enzymes correlated with benzoate depletion, as did induction of catabolism of alternative substrates, both endogenous (lipids, carbohydrates, and proteins) and exogenous. Thus, we detected a transient 5-fold abundance increase for phthalate, phthalate ester, biphenyl, and ethyl benzene catabolic enzymes, which coincided with at least 4-fold increases in phthalate and biphenyl catabolic activities. Stationary-phase cells demonstrated an ∼250-fold increase in carbon monoxide dehydrogenase (CODH) concurrent with a 130-fold increase in CODH activity, suggesting a switch to CO or CO(2) utilization. We observed two phases of stress response: an initial response occurred during the transition to stationary phase, and a second response occurred after the cells had attained stationary phase. Although SigG synthesis was induced during starvation, a ΔsigG deletion mutant showed only minor changes in cell survival. Stationary-phase cells underwent reductive cell division. The extreme capacity of RHA1 to survive starvation does not appear to involve novel mechanisms; rather, it seems to be due to the coordinated combination of earlier-described mechanisms.

Development of novel rat model for high-fat and high-cholesterol diet-induced steatohepatitis and severe fibrosis progression in SHRSP5/Dmcr.

OBJECTIVES: Patients with nonalcoholic fatty liver disease are increasing worldwide, and preventive measures are an urgent need and primary concern today. AIM: This study aimed to develop and clarify the usefulness of the SHRSP5/Dmcr rat, derived from a stroke-prone spontaneously hypertensive rat, as a novel animal model for time-course analysis of steatohepatitis and the severe fibrosis progression often observed in the disease. METHODS: Ten-week-old male SHRSP5/Dmcr rats were divided into six groups: half were fed a high-fat and high-cholesterol-containing diet (HFC diet), and the others the control, stroke-prone (SP) diet for 2, 8, and 14 weeks. RESULTS: The HFC diet significantly increased serum transaminase and gamma glutamyl transpeptidase activities, tumor necrosis factor alpha levels, and serum and hepatic total cholesterol levels over time. In contrast, this diet decreased serum albumin, glucose, and adiponectin levels throughout or the later stage of the feeding period, but did not influence serum insulin levels. Histopathologically, the HFC diet increased microvesicular steatosis, and focal or spotty necrosis with lymphocyte infiltrations were observed in the liver at 2 weeks, macrovesicular steatosis, ballooned hepatocytes with Mallory-Denk body formation in some, and multilobular necrosis and fibrosis at 8 weeks. Interestingly, this fibrosis formed a honeycomb network at 14 weeks. These changes are very similar to those observed in patients with non-alcoholic steatohepatitis. CONCLUSIONS: SHRSP5/Dmcr rats appear to be a useful model for analyzing the time-dependent changes of HFC diet-induced steatohepatitis and fibrosis progression.

Rapeseed (canola) oil aggravates metabolic syndrome-like conditions in male but not in female stroke-prone spontaneously hypertensive rats (SHRSP).

This study was conducted to investigate whether or not there are sex differences in canola oil (CAN)-induced adverse events in the rat and to understand the involvement and the role of testosterone in those events, including life-shortening. Stroke-prone spontaneously hypertensive rats (SHRSP) of both sexes were fed a diet containing 10 wt/wt% soybean oil (SOY, control) or CAN as the sole dietary fat. The survival of the males fed the CAN diet was significantly shorter than that of those fed the SOY diet. In contrast, the survival of the females was not affected by CAN. The males fed the CAN diet showed elevated blood pressure, thrombopenia and insulin-tolerance, which are major symptoms of metabolic syndrome, whereas such changes by the CAN diet were not found in the females. Plasma testosterone was significantly lower in animals of both sexes fed the CAN diet than in those fed the SOY diet, but interestingly, the lowered testosterone was accompanied by a marked increase in plasma aldosterone only in the males. These results demonstrate significant sex differences in CAN-toxicity and suggest that those sex differences may be attributable to the increased aldosterone level, which triggers aggravation of the genetic diseases specific to SHRSP, that is, metabolic syndrome-like conditions, but only in the males. The present results also suggest that testosterone may negatively regulate aldosterone production in the physiology of the males, and the inhibition of that negative regulation caused by the CAN diet is one of the possible causes of the adverse events.

CT of the pancreas: comparison of anatomic structure depiction, image quality, and radiation exposure between 320-detector volumetric images and 64-detector helical images.

PURPOSE: To prospectively compare 320-detector volumetric and 64-detector helical computed tomographic (CT) images of the pancreas for depiction of anatomic structures, image noise, and radiation exposure. MATERIALS AND METHODS: This study was approved by the institutional review board, and written informed consent was obtained. A total of 154 patients (85 men, 69 women; age range, 26-85 years; mean age, 67 years) who underwent biphasic (arterial and pancreatic phase) contrast material-enhanced CT performed with a 320-detector scanner were randomized into two groups: the 320-detector group and the 64-detector group. Biphasic transaxial multiplanar reformatted images and volume-rendered CT angiograms were obtained. CT numbers in the abdominal aorta, pancreas, and abdominal wall fat tissue; signal-to-noise ratio (SNR); and dose-length product (DLP) were compared. In addition, image quality and focal lesion depiction (n = 35) were qualitatively determined in the two groups. Unpaired t and Mann-Whitney tests were used for quantitative and qualitative assessment, respectively. RESULTS: No significant difference in CT numbers of the abdominal aorta and pancreas was noted between the two groups. Mean DLP was 43% lower in the 320-detector group (675.4 mGy·cm) than in the 64-detector group (1187.8 mGy·cm) (P < .001). SNR of the abdominal aorta, pancreas, and abdominal wall fat on biphasic images was significantly lower in the 320-detector group than in the 64-detector group (P < .001). Image quality was acceptable in both groups and was slightly better in the 64-detector group for pancreatic phase axial images (P = .02) and arterial phase multiplanar reformatted images (P < .01). No significant difference was found in the depiction of pancreatic parenchyma, main pancreatic duct, focal pancreatic lesions, splanchnic arteries, or most of the small splanchnic arterial branches. CONCLUSION: A 320-detector CT scan facilitates fast volumetric contrast-enhanced CT of the entire pancreas with acceptable image quality, even though SNR was significantly lower at 320-detector volumetric scanning.

Idarubicin, an Anthracycline, Induces Oxidative DNA Damage in the Presence of Copper (II).

BACKGROUND/AIM: The aim of the present study was to investigate whether idarubicin (IDR) induces oxidative DNA damage in the presence of copper (II). MATERIALS AND METHODS: DNA damage was evaluated by pBR322 plasmid DNA cleavage. The formation of oxidative stress markers [O2 •- and 8-hydroxy-2'-deoxyguanosine (8-OHdG)] was analysed. RESULTS: IDR induced DNA damage and O2 •- and 8-OHdG generation in the presence of copper (II). CONCLUSION: IDR induced oxidative DNA damage in the presence of copper (II). Since it has been reported that the concentration of copper in the serum of cancer patients is higher than that in healthy groups, IDR-induced oxidative DNA damage in the presence of copper (II) may play an important role in anticancer therapeutic strategies.

Dietary soybean oil, canola oil and partially-hydrogenated soybean oil affect testicular tissue and steroid hormone levels differently in the miniature pig.

The present study was conducted to examine the influence of dietary canola oil (CAN) and partially-hydrogenated soybean oil (HSO) compared to soybean oil (SOY, control) on the morphology and function of testes using miniature pigs as the test subject. Male miniature pigs were fed a diet containing 10%SOY, 9%CAN+1%SOY, or 9%HSO+1%SOY for 18 months. The scheduled autopsies revealed no abnormalities in histopathological examination of the major organs, except the testes. Atrophy of the seminiferous tubules and hyperplasia in the Leydig cells were found in the SOY and CAN groups. DNA microarray analysis indicated downregulation in the CAN and the HSO groups of genes encoding for gonadotropins in the pituitary gland and of enzymes and proteins involved in steroid hormone metabolism in the testes, compared to the SOY group. Plasma levels of sex hormones in the CAN and HSO groups tended to be higher and testosterone and dihydrotestosteorne in the HSO group were significantly higher than in the SOY group. These results demonstrate that testes are morphologically and functionally affected by the dietary oils, while the plasma steroid hormone levels do not necessarily reflect the gene expression, probably owing to feedback regulation via the gonadal hormones in the hypothalamus-pituitary-gonadal axis.

Isolation and Characterization of Genes Responsible for Naphthalene Degradation from Thermophilic Naphthalene Degrader, Geobacillus sp. JF8.

Geobacillus sp. JF8 is a thermophilic biphenyl and naphthalene degrader. To identify the naphthalene degradation genes, cis-naphthalene dihydrodiol dehydrogenase was purified from naphthalene-grown cells, and its N-terminal amino acid sequence was determined. Using a DNA probe encoding the N-terminal region of the dehydrogenase, a 10-kb DNA fragment was isolated. Upstream of nahB, a gene for dehydrogenase, there were two open reading frames which were designated as nahAc and nahAd, respectively. The products of nahAc and nahAd were predicted to be alpha and beta subunit of ring-hydroxylating dioxygenases, respectively. Phylogenetic analysis of amino acid sequences of NahB indicated that it did not belong to the cis-dihydrodiol dehydrogenase group that includes those of classical naphthalene degradation pathways. Downstream of nahB, four open reading frames were found, and their products were predicted as meta-cleavage product hydrolase, monooxygenase, dehydrogenase, and gentisate 1,2-dioxygenase, respectively. A reverse transcriptase-PCR analysis showed that transcription of nahAcAd was induced by naphthalene. These findings indicate that we successfully identified genes involved in the upper pathway of naphthalene degradation from a thermophilic bacterium.

Oxidative DNA Damage and Apoptosis Induced by Aclarubicin, an Anthracycline: Role of Hydrogen Peroxide and Copper.

BACKGROUND/AIM: This study aimed to investigate aclarubicin (ACR)-induced oxidative DNA damage and apoptosis. MATERIALS AND METHODS: ACR-induced apoptosis was analyzed using HL-60 leukemia cells and HP100 cells, hydrogen peroxide (H2O2)-resistant cells derived from HL-60 cells. ACR-induced DNA damage was analyzed using plasmid DNA. RESULTS: HL-60 cells were more sensitive to ACR than HP100 cells. In HP100 cells, DNA ladder formation and caspase-3/7 activity induced by ACR were suppressed or delayed in comparison to those in HL-60 cells. ACR-induced DNA damage occurred in the presence of Cu(II), and scavenger experiments showed that the reactive species causing DNA damage appeared to be generated from H2O2 and Cu(I). Moreover, we detected intracellular Cu(I) induced by ACR in HL-60 cells, using CopperGREEN™, a fluorescent probe for detection of Cu(I) ion specifically. CONCLUSION: ACR-induced DNA damage and apoptosis can be accounted for by the involvement of H2O2 and Cu(I).

Oxidative DNA Damage Induced by Pirarubicin, an Anthracycline Anticancer Agent, in the Presence of Copper(II).

BACKGROUND/AIM: One mechanism of the anticancer action of anthracyclines is believed to be oxidative DNA damage. Previously, we reported that doxorubicin induced oxidative DNA damage in the presence of Cu(II). However, the mechanism of pirarubicin-induced oxidative DNA damage has not been well clarified. MATERIALS AND METHODS: DNA damage by pirarubicin in the presence of Cu(II) was analyzed using pBR322 plasmid DNA. O2•- derived from pirarubicin in the presence of Cu(II) was detected by cytochrome c reduction. RESULTS: Pirarubicin induced DNA damage in the presence of Cu(II). Scavenger experiments suggest that reactive species are generated from H2O2 and Cu(I). Pirarubicin induced O2•- production in the presence of Cu(II). CONCLUSION: These findings suggest that pirarubicin plus Cu(II) induces oxidative DNA damage in a similar manner to doxorubicin, and Cu(II)-mediated oxidative DNA damage may serve as a common mechanism for antitumor effects of anthracyclines.

Dietary lipids impacts on healthy ageing.

Healthy ageing is gaining attention in the lipid nutrition field. As in vivo biomarkers of healthy ageing, we have evaluated the survival, learning/memory performance, and physical potencies in rodents fed a diet supplemented with high-linoleic acid (LNA, omega6) safflower oil or high-alpha-linolenic acid (ALA, omega3) perilla oil for long periods. The results suggested that perilla oil with a low omega6/omega3 ratio is beneficial for healthy ageing. In order to address this issue further, we determined the survival of stroke-prone SHR (SHRSP) rats fed a conventional rodent diet supplemented with 10% fat or oil. Survival was longer with omega3-rich oils compared with omega6-rich oils. However, some kinds of vegetable oils and hydrogenated oils shortened the survival of SHRSP rats to an unusual degree (ca. 40% compared with that of omega6-rich oil) that could not be accounted for by the fatty acid and phytosterol composition of the oils. The observed decrease in platelet counts was associated with pathological changes in the kidney and other organs. Dihydro-vitamin K1 is proposed as a likely candidate as a stroke-stimulating factor in hydrogenated oils. Thus, factors other than fatty acids (omega6/omega3 balance) and phytosterols must be taken into account when fats and oils are evaluated in relation to healthy ageing.